Calmodulin (CaM) is found in all eucaryotic cells where it plays a key role in orchestrating Ca 2+-dependent events in the cell by virtue of its ability to modulate the activities of a number of different enzyme targets. Among these are several protein kinases, phosphodiesterase, adenylate cyclase, inositol 1,4,5-trisphosphate 3-kinase, and nitric oxide synthase. Our long-term goal is to define the structural determinants that are necessary for in vitro binding and activation of different target enzymes by CaM. CaM is a dumbbell shaped molecule. The lobes or "weights" of the dumbbell each consist of a pair of EF hand Ca2+-binding domains. In spite of structural similarities between the lobes of CaM, each can perform unique functions in CaM-target complexes. It is our hypothesis that many functional differences between the CaM lobes are associated with unique amino acid determinants; i.e., determinants at homologous positions in the lobes that are not functionally interchangeable. We have three basic short- to medium-term goals. (1) To identify positions in the lobes containing unique determinants for binding and activation of enzyme targets. (2) To identify unique determinants associated with correct binding of peptides representing the isolated CaM-binding domains of target enzymes. (3) To distinguish unique determinants for direct interactions between CaM and the CaM-binding domain in different target enzymes from those for interactions between CaM and other regions in the targets. The specific experimental aims are: (1) Synthesis of altered CaMs in which residues at homologous positions in the lobes have been swapped for one another using recombinant DNA techniques. (2) Initial verification of the main-chain conformations of altered CaMs by measuring the Ca2+-binding affinities and stoichiometries for each protein and by evaluating their CD spectra in the presence and absence of Ca2+. Proteins failing initial verification will not be studied further. (3) Determination of the abilities of altered CaMs to bind and activate myosin light chain kinase, plant NAD kinase, calcineurin, and nitric oxide synthetase activities. (4) Determination of the crystal structures for each altered CaM as final verification of its expected three dimensional structure. (5) Determination of a dissociation constant for the complex between each of the altered CaMs and a peptide representing the CaM-binding domain of MLCK. (6) Characterization of the spatial relationship between CaM and the peptide in (5) by estimating distances between sites on bound peptide and those on native or altered CaMs using fluorescence energy transfer techniques.